CN1231114A - Protoplast cultivation for Chinese cabbage and regeneration planting method - Google Patents
Protoplast cultivation for Chinese cabbage and regeneration planting method Download PDFInfo
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Abstract
The protoplast culture of loose-head Chinese cabbage and plant regeneration method includes the following steps: collecting the hypocotyl of loose-head Chinese cabbage Ogu CMS with 5d seedling age or the cotyledon with 14-18d seedling age, freely immersing the above-mentioned material in enzyme liquor formed from 1.0-2.0% of cellulase, 0.1-1.0% of pectolase, 10 mM CaCl2.2H2O, 0.7 mM KH2PO4 and 0.5M mannite for 25-60 hr, centrifugal collecting protoplast at 500 rpm, culturing the protoplast in the culture medium formed from KM8P, +2,4-D (0.5mg/L)+6-BA (0.25 mg/L)+NAA(0.5-1.0mg/L) for 3 weeds to form small calli, buds differentiation in the culture medium formed from MS+6-BA (10.0 mg/L)+NAA (0.3 mg/L), and making the above-mentioned differentiated buds root in the MS culture medium for 2 weeks to obtain the complete regenerated plant. Its plant regeneration rate is 1.8%.
Description
The invention belongs to plant protoplast and cultivate and plant regeneration method, be exclusively used in the Chinese cabbage protoplast and cultivate and plant regeneration.
Brassicaceous vegetable has very significantly hybrid vigour, and utilizing the cytoplasm male sterility line production of hybrid seeds then is the most economical and effective fundamental way of heterosis utilization, so each state of the world all payes attention to male sterile research of pair cell matter and utilization especially.Over nearly 20 years, on crop in cruciferae, find and cultivate the cytoplasmic male sterility type of multiple separate sources both at home and abroad, and research is maximum, utilization is Ogu CMS (male sterile with radish cytoplasm) and PolCMS (pol cytoplasmic male sterility (pol cms)) the most widely.Chinese cabbage (Brassica campestrisssp.chinensis Makino) originates in China, and in recent years, states such as Southeast Asia, Japan and the United States and Europe extensively introduce a fine variety, and oneself becomes worldwide vegetables gradually.My school Vegetable Research Institute is since the mid-1970s, seed selection and utilize male sterile two-purpose line to carry out the production of hybrid seeds at home and abroad takes the lead in, and successively breed " short assorted ", " short anti-" serial good hybrid large scale application in production, but dual-purpose be breeding method pull out can educate strain not only take a lot of work, time-consuming, increase cost, and be difficult for guaranteeing hybrid purity.Utilize Chinese cabbage cytoplasmic male sterile line (CMS) then can overcome these shortcomings.Event cultivates to study to its protoplast has important theory and practice significance.In brassica plant, after Kartha etc. (1974) cultivate rape mesophyll protoplast regenerated plant first, just on wild cabbage, leaf mustard, cabbage mustard, Chinese cabbage, make a breakthrough up to the mid-80, culture technique is gradually improved, but Chinese cabbage mesophyll, hypocotyl and root protoplast are cultivated and all failed regeneration plant.The Ogura sterile source is the male sterile individuality (the performance bud is little, style bending, stamen flapization) that little storehouse was found in radish varieties seed field of Japan Kagoshima in nineteen sixty-eight, after seed selection forms male sterile line.By a large amount of evidences, its sterility is to be controlled jointly by plasmon and 2 pairs of recessive karyon genes, but does not have [fertility.Because of the Latin language in little storehouse is Ogura, so the radish cytoplasm sterile source is called Ogu CMS source.Ogu CMS now spreads throughout the world, is used for the male sterile utilization research of crop in cruciferae.
The objective of the invention is to the defective that exists in the above-mentioned prior art, provide a kind of Chinese cabbage protoplast to cultivate and plant regeneration method, making that Chinese cabbage mesophyll and hypocotyl protoplast are cultivated can regeneration plant.
Chinese cabbage protoplast provided by the present invention is cultivated and the regeneration plant method, and its concrete steps are as follows:
1) gets the Chinese cabbage Ogu CMS hypocotyl of 5d seedling age or the cotyledon of 14～18d seedling age, at 1.0～2.0% cellulases+0.1～1.0% pectase+10mMCaCl
22H
2O+0.7mMKH
2PO
4Free 25～60hr in the enzyme liquid of+0.5M mannitol, centrifugal collection protoplast under the 500rpm;
2) protoplast is at KM8P
1+ 2, on the medium of 4-D0.5mg/L+6-BA0.25mg/L+NAA0.5～1.0mg/L, cultivate and 3 weeks formed small callus;
3) through MS+2,4-D1.0mg/L breeds callus;
4) be to break up bud on the MS+6-BA10.0mg/L+NAA0.3mg/L at medium.
5) above-mentioned differentiation bud is taken root on the MS medium, can obtain again complete after 2 weeks
Living plant.
Chinese cabbage protoplast provided by the present invention is cultivated and plant regeneration method, and shoot regeneration frequency is 1.8%.Regeneration plant moves in the nutritive cube through the domestication hardening, and the sleeving plastic bag preserves moisture, and removes plastic sack after the week, cultivates about 15d again, is transplanted to allow it fully grow, budding, bloom in the big flowerpot.In recent years, rape belongs to cabbage heart, purple flowering stalk, cauliflower in the rape kind, all obtains regeneration plant from protoplast, but never success in common Chinese cabbage, so be considered in the rape genus rape kind one of plant of the most difficult living plant.The present invention at home and abroad reported first Chinese cabbage cotyledon protoplast and OguCMS hypocotyl protoplast cultivate all can regeneration plant.According to experimental result provided by the present invention, seedling age has the greatest impact to it in the incubation of hypocotyl protoplast; Secondly, the growth hormone and the basic element of cell division must make up by a certain percentage, improper division and the growth that is unfavorable for cell of ratio.
Embodiment 1: the Chinese cabbage cotyledon protoplast is cultivated
1) be 91H autumn-21 (maintenance lines) for the examination material, the flowing water flushing 10～20min of seed elder generation, the alcohol disinfecting 1min with 70% is at 0.1%HgCl
2After soaking 30min in the solution, use aseptic washing 5 times again, sowing is germinateed under 25 ℃ of constant temperatures on the MS medium.
2) get the cotyledon of 14～18d seedling age, be cut into the wide slice of 2mm, place 5 kinds of enzyme liquid, under 25 ℃ of dark conditions, through 26,36,46,56hr digestion, protoplast suspension filters with 50 μ nylon mesh, centrifugal 15min (500rpm), abandon supernatant after, add CPW washing lotion centrifuge washing 3 times, the protoplast of purifying suspends with improvement KM8P medium, and with the dyeing of 0.1% phenol safflower, measures the protoplast vigor.
3) cotyledon protoplast is cultivated and to be established 6 kinds of medium: 1. MS; 2. B
53. DPD; 4. mKM8P; 5. DPD
1: DPD medium--the B of improvement
5Organic+2,4-D0.1mg/L+6-BA0.5mg/L; 6. KM8P
1: KM8P medium--the B of improvement
5Trace element+KM8P organic component, organic acid, sugar alcohol+glucose 9.0%+ sucrose 1.0%+ galactose 0.03%+2,4-D0.5mg/L+NAA0.5mg/L+6-BA0.25mg/L; 1.～5. medium, mannitol is 0.45M; 1.～4. medium all adds 2,4-D0.1mg/L+6-BA1.0mg/L.Carry out embedding (adding 1.2% low melting point agarose) and shallow-layer liquid culture respectively, the protoplast culture density is 1～1.5 * 10
5Individual/ml.
4) protoplast is cultivated under dark condition earlier, forwards under the low light level to cultivate again, and adds a small amount of fresh culture (totally 3 times, mannitol content drops to 0.3M, 0.15M, 0M successively) behind the 8d.After cultivation the 5th, 10,15 and 20d statistics division frequency.
5) with little callus transfer of granules of 1～2mm size to MS+2,4-D1.0mg/L and MS+2, propagation forms behind the callus to MS+6-BA10.0mg/L+NAA0.1mg/L on the 4-D0.2mg/L+KT0.5mg/L solid culture medium; MS+ZT1.0mg/L+GA
30.1mg/L and induced bud differentiation on the MS+Zea10.0mg/L+NAA0.1mg/L medium.
6) bud with elongation forwards on the MS medium of no hormone, takes root after 2 weeks, thereby obtains complete regeneration plant.Embodiment 2:0guCMS hypocotyl protoplast is cultivated
1) through how the OguCMS (91H autumn-100) for backcross transformation is the examination material (BC7) to adopt Chinese cabbage seminar of department of horticulture of Agricultural University Of Nanjing, behind the aseptic seeding, the dark cultivation, get 3,5,7,9,11,13, (the earlier vertical partial application of the hypocotyl of 16d seedling age, be cut into the long section of 2mm again) separate protoplast, with 2.0% cellulase+1.0% pectase+10mMCaCl
22H
2O, 0.7mMKH
2PO
4The enzyme liquid of+0.5M mannitol protoplast that dissociates, the cleaning of protoplast and collect all centrifugal with 500rpm.
2) KM8P in improvement adds 2, on the 4-D0.5mg/L+NAA1.0mg/L+6-BA0.25mg/L medium, cultivates and 3 weeks promptly forms macroscopic callus, can reach the 1mm size about 5 weeks.
3) at MS+2, breed callus on the 4-D1.0mg/L medium, the bud differential medium is MS+6-BA10.0mg/L+NAA0.3mg/L.
4) root media is MS.Result of implementation is as follows: 1. protoplasm free
1) influence of enzyme liquid concentration: as can be seen from Table 1,1.0% cellulase+0.1% pectinase enzymatic hydrolysis 46hr, cotyledon protoplast output and division frequency are all the highest, and brownization and percentage of damage are then lower.The processing of 0.8% cellulase+0.1% pectase, every index occupy the 2nd.In the processing of cellulase 1.5%+ pectase 0.2% and cellulase 1.0%+ pectase 1.0%, though go the wall rate to reach 100% fully, brownization and broken more is so output and division frequency obviously reduce.
2) influence of enzymolysis time: under the enzyme liquid concentration conditions of 1.0% cellulase and 0.1% pectase, the free time of cotyledon protoplast and OguCMS hypocotyl protoplast is good with 46hr and 36hr respectively, performance output height goes wall to reach the high comprehensive excellent results of survival rate fully.
3) seedling age is to the influence of OguCMS hypocotyl protoplasm free: the result shows, the effect of separating protoplast with the hypocotyl of 5d seedling age is best, and its output is 2～3 * 10
6Individual/g, the division frequency behind the cultivation 10d is 11.5%, all reaches the highest; Seedling age 3d takes second place, and is respectively 1～2 * 10
6Individual/g and 8.0%; Behind the seedling age 7d, then significantly reduce, be respectively 3～4 * 10
3Individual/g and 3.5%; Seedling age then reduces to 3.0 * 10 respectively after surpassing 9d
2Individual/g and 0.0% can not be as the material that separates protoplast.
4) influence of centrifugation rate; Centrifugal under 1500rpm, protoplast breaks in a large number, and output is not high, and under the 1000rpm speed, the protoplast that breaks obviously reduces, and output increases (4 * 10
4Individual/g); And under the 500rpm condition, no matter be that cotyledon or the fragmentation of hypocotyl protoplast are all less, collecting effect is good, and output can reach 10
4～10
6Individual/g.2. protoplast cultivates 1) the cotyledon protoplast cultivation: the result shows, under the suitable culture condition, the Chinese cabbage cotyledon protoplast is cultivated 1～2d and is promptly begun first division, 5～6d second division, very fast formation small cell cluster, 20d has formed spherical or sternzellen group, promptly forms macroscopic small callus after 1 month.From the influence (table 2) of different medium to culture effect, KM8P
1Protoplast division frequency height not only, and do not have brownization; MKM8P medium, the protoplast division frequency behind the 20d are 38.0% and slight brownization are arranged; MS, B
5, DPD, DPD
14 kinds of medium cultivate that the protoplast division frequency is 5.0～11.5% behind the 20d, and brownization degree is medium～and serious; As seen, the KM8P of improvement
1Medium is suitable for the cultivation of Chinese cabbage cotyledon protoplast most.2) OguCMS hypocotyl protoplast is cultivated: in 12 HORMONE TREATMENT combinations, and hypocotyl protoplast division performance different (table 3), 6-BA0.25mg/L+NAA1.0mg/L hypocotyl protoplast division is the most vigorous; 6-BA0.5mg/L+NAA1.0mg/L protoplast also has higher division frequency; 6-BA0.25mg/L and NAA is respectively 0.5,1.5, the combination of 2.0mg/L and the combination of 6-BA0.50mg/L+NAA0.5mg/L, the protoplast division frequency is low; 6-BA0.5mg/L and NAA is respectively 1.5, the combination of 2.0mg/L and the combination of 6-BA1.0mg/L+NAA0.5mg/L, the protoplast division frequency is extremely low; The all acellular division of other 3 treatment combination OguCMS hypocotyl protoplasts.The hypocotyl protoplast is cultivated, and begins division about 48hr, can see 2～3 divisions about 5d.The hypocotyl protoplast also will form several normal colonies in cultivation, the person then easily sinks to the bottom, breaks or brownization not form the colony, so that can not form cell mass.OguCMS hypocotyl protoplast is at KM8P
1Additional 6-BA0.25mg/L+NAA1.0mg/L+2, the new wall of regeneration is fast with the division startup on the medium of 4-D0.5mg/L, and division frequency all is higher than other combination, is the optimal medium that carries out the cultivation of OguCMS hypocotyl protoplast.3. the proliferation test of callus shows, the callus that the Chinese cabbage cotyledon protoplast forms is at MS+2, and growth rapidly on the 4-D0.2mg/L+KT0.5mg/L medium, compact structure, and the green point of tool, these green points easily form green callus, and organs such as easy differentiation is sprouted, leaf.The callus that OguCMS hypocotyl protoplast forms is then at MS+2,4-D1.0mg/L cultivation effect is good on the medium, this shows 2,4-D is very important to the propagation of callus, is not containing 2,4-D or contain higher concentration (1.5～2.0mg/L), low concentration is (on 0.2～0.5mg/L) the medium, callus Growth is slow, so in the OguCMS hypocotyl protoplast callus multiplicative stage, must strictly control 2, the concentration of 4-D is 1.0mg/L.4. indefinite bud forms and plant regeneration
Callus promptly forms indefinite bud about 20d on differential medium, but the callus of hypocotyl protoplast is than the difficult indefinite bud that forms of the callus of cotyledon protoplast.The cotyledon protoplast callus is at MS+6-BA10.0mg/L+NAA0.1mg/L, MS+KT1.0mg/L+GA
3O.1mg/L, on the differential medium of MS+Zea10.0mg/L+NAA0.1mg/L, all can differentiate indefinite bud, differentiation rate is respectively 11.5%, 6.5%, 8.0%, on average breaks up the bud number and is respectively 1.3,1.0,1.0.Hypocotyl protoplast callus is on 10 kinds of differentiation culture (table 4), and the differentiation adventitious buds rate is very low, and differentiation be simple bud.6-BA10.0mg/L+AAA0.3mg/L the bud differentiation rate of medium is the highest, also has only 5.5%; 6-BA10.0mg/L with NAA0.2,0.4 combination and the medium of 6-BA55.0mg/L+NAA0.3mg/L, the differentiation adventitious buds rate is respectively 1.0%, 3.0%, 0.8%, other 6 treatment combinations all do not break up bud.
With the indefinite bud of cotyledon protoplast and the formation of OguCMS hypocotyl protoplast, be seeded in the MS cultivation that does not contain any hormone and take root, between the two no significant difference.Take root after 2 weeks, can obtain whole plant, shoot regeneration frequency is 1.8%.Regeneration plant moves in the nutritive cube through the domestication hardening, and the sleeving plastic bag preserves moisture, and removes plastic sack after the week, cultivates about 15d again, is transplanted to allow it fully grow, budding, bloom in the big flowerpot.
Table 1 enzyme liquid concentration is gone (%) (number/g leaf) rate (%) rate (%) rate (%) 1.5 0.2 2.5 * 10 of wall division brownization of frequency and broken enzyme (%) fully to the cellulose pectase protoplast output that influences of the free effect of Chinese cabbage cotyledon protoplast
4100 8.5 30.01.0 1.0 2.0 * 10
4100 3.5 33.01.0 0.5 2.5 * 10
478 13.0 21.51.0 0.1 2.0 * 10
596 14.5 6.50.8 0.1 1.5 * 10
594 11.5 5.0
Annotate: 5 kinds of all additional 10mMCaCl of enzyme liquid
22H
2O+0.7mMKH
2PO
4+ 0.5M mannitol; The time 46hr that dissociates, centrifugation rate 500rprm; Protoplast output repeats 3 times with " blood counting chamber " statistics; Division frequency, brownization rate are added up after cultivating 15d.
The culture effect of table 2 Chinese cabbage cotyledon protoplast on different medium
Annotate: the shallow-layer liquid culture;-be no brownization ,+slight brownization, ++ moderate brownization, +++serious brownization.
| Medium | Division frequency (%) | Brownization degree | |||
| ??5d | ??10d | ???15d | ???20d | ||
| ???MS ???B 5???DPD ???DPD 1???mKM8P ???KM8P 1 | ??0 ??0.5 ??1.5 ??0 ??2.0 ??5.0 | ??1.5 ??3.0 ??5.0 ??1.0 ??5?5 ??12.5 | ???3.0 ???4.5 ???8.5 ???3.0 ???18.5 ???28.5 | ???5.5 ???7.5 ???11.5 ???5.0 ???38.0 ???53.5 | ????+++ ????+++ ????++ ????+++ ????+ ????- |
The influence that the different HORMONE TREATMENT combinations of table 3 are cultivated OguCMS hypocotyl protoplast
| ????6-BA(mg/L) | ?????????????????NAA(mg/L) | |||
| ????0.5 | ???1.0 | ????1.5 | ???????2.0 | |
| ????0.25 ????0.50 ????1.0 | ????++ ????++ ????- | ???++++ ???+++ ???- | ????++ ????+ ????- | ???????++ ???????+ ???????- |
Annotate: minimal medium is KM8P
1, all add 2,4-D0.5mg/L.-. there is not division; + .2～5 cell, division frequency is extremely low; ++ .6～10 cells, division frequency is low; +++.11～15 cell, division frequency height; ++ ++ .16～20 cell, division frequency is high.Cultivate back 20d investigation.
Table 4 6-BA and NAA concentration are to OguCMS hypocotyl protoplast callus
Indefinite bud forms influences differentiation rate (%)
| ???6-BA ??(mg/L) | ???????????????????????NAA(mg/L) | ||||
| ???0.1 | ????0.2 | ???0.3 | ????0.4 | ????0.5 | |
| ???10.0 ???5.0 | ????0 ????0 | ????1.0 ????0 | ???5.5 ???0.8 | ????3.0 ????0 | ?????0 ?????0 |
Annotate: minimal medium is MS.
Claims (2)
1. Chinese cabbage protoplast cultured method, concrete steps are as follows:
1) gets the Chinese cabbage Ogu CMS hypocotyl of 5d seedling age or the cotyledon of 14～18d seedling age, at 1.0～2.0% cellulases+0.1～1.0% pectase+10mMCaCl
22H
2O+0.7mMKH
2PO
4Free 25～60hr in the enzyme liquid of+0.5M mannitol, centrifugal collection protoplast under the 500rpm;
2) protoplast is at KM8P
1+ 2, on the medium of 4-D0.5mg/L+6-BA0.25mg/L+NAA0.5～1.0mg/L, cultivate and 3 weeks formed small callus;
3) through MS+2.4-D1.0mg/L propagation callus;
4) be that MS+6-BA10.0mg/L+N forms indefinite bud from 0.3mg/L at medium.
2. the regeneration plant method of the described Chinese cabbage protoplast cultivation of claim 1 is that above-mentioned indefinite bud is taken root on the MS medium, can obtain complete regeneration plant after 2 weeks.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99114127A CN1108096C (en) | 1999-03-29 | 1999-03-29 | Protoplast cultivation for Chinese cabbage and regeneration planting method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99114127A CN1108096C (en) | 1999-03-29 | 1999-03-29 | Protoplast cultivation for Chinese cabbage and regeneration planting method |
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| Publication Number | Publication Date |
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| CN1231114A true CN1231114A (en) | 1999-10-13 |
| CN1108096C CN1108096C (en) | 2003-05-14 |
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ID=5277245
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388803A (en) * | 2011-08-12 | 2012-03-28 | 江苏省农业科学院 | Chilli cytoplasm male sterile line protoplast separation purification and callus forming method |
| CN116941532A (en) * | 2023-08-29 | 2023-10-27 | 安徽江淮园艺种业股份有限公司 | Method for promoting regeneration of protoplast by using low-temperature plasma |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144517C (en) * | 1999-03-29 | 2004-04-07 | 南京农业大学 | Asymmetrical cytomixis method for leaf-unrolling Chinese cabbage and the regenerated plant obtained thereby |
-
1999
- 1999-03-29 CN CN99114127A patent/CN1108096C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388803A (en) * | 2011-08-12 | 2012-03-28 | 江苏省农业科学院 | Chilli cytoplasm male sterile line protoplast separation purification and callus forming method |
| CN116941532A (en) * | 2023-08-29 | 2023-10-27 | 安徽江淮园艺种业股份有限公司 | Method for promoting regeneration of protoplast by using low-temperature plasma |
| CN116941532B (en) * | 2023-08-29 | 2024-04-16 | 安徽江淮园艺种业股份有限公司 | Method for promoting regeneration of protoplast by using low-temperature plasma |
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